Haemophilus influenzae type b (Hib) is a major cause of bacterial meningitis in children under five years of age (refs. 1, 2. The literature references are identified at the end of this disclosure). The bacterium is protected from phagocytosis by a polysaccharide capsule that is a repeating polymer of polyribosyl ribitol phosphate (PRP). Antibodies induced against the capsular polysaccharide of the organism are protective (ref. 3). Effective conjugate vaccines in which PRP is linked to different carrier proteins such as diphtheria toxoid (PRP-D), tetanus toxoid (PRP-T), CRM 197 (HbOC) and the outer membrane protein of Neisseria meninaitidis have been developed (refs. 4, 5). However, these conjugate vaccines do not protect against other invasive encapsulated H. influenzae type a and c strains and, more importantly, against non-encapsulated non-typeable H. influenzae strains that are one of the common causes of otitis media for which there is no vaccine. Therefore, the inclusion of selected non-encapsulated H. influenzae immunogens in current Hib vaccines is necessary to develop a universal Hi vaccine.
Granoff and Munson (ref. 6) have reported that antibodies directed against Hib outer membrane proteins (OMP) P1, P2 and P6 were protective in the infant rat model of bacteremia. Therefore, a promising strategy for designing a universal H. influenzae vaccine with enhanced protective ability would be to use either purified OMPs or their protective epitopes as additional immunogens and carriers for PRP. The gene coding for P1 has been cloned from several different Hib subtypes (refs. 7, 8). The comparative analysis of P1 protein sequences from these Hib isolates revealed the existence of three hypervariable regions. Indeed, the P1-specific MAbs reported by Hansen's group recognize only 50% of the Hib isolates tested (refs. 7, 9). For the P2 protein, although the nucleotide sequences of the P2 gene isolated from two different Hib subtypes (1H and 3L) were found to be identical (refs. 10, 11), some amino acid variability was found among the P2 sequences of two other Hib subtypes (2L and 6U) (ref. 11). In contrast, analysis of antigenic determinants, gene sequences and restriction fragment length polymorphisms experiments indicated that the P6 protein was highly conserved among all strains of Hi (ref. 12).
Recent studies showed that a murine P1-specific monoclonal antibody (MAb 7C8) and rabbit antisera raised against purified P1 from either typeable or non-typeable H. influenzae strains were protective in animal models (refs. 9, 13, 14). Murphy and Bartos (ref. 15) also reported that a monoclonal antibody recognizing a surface-exposed epitope of a non-typeable H. influenzae P2 protein had bactericidal activity in vitro. Anti-P1 and anti-P2 monoclonal antibodies were found to cross-react with typeable and non-typeable strains of H. influenzae (refs. 16 to 18). However, there are still serious concerns with the use of whole native Hib OMPs as an efficacious universal vaccine against both typeable and non-typeable Hi. Firstly, children who recover from otitis media caused by non-typeable Hi generally develop bactericidal antibodies against variable antigens, such as P2 and lipooligosaccharides. Secondly, the P1 and P2 cross-protective epitopes described above have not yet been identified. Thirdly, it was reported (ref. 12) that the epitope(s) recognized by anti-P6 bactericidal antibodies are expressed in small amounts on the bacterial surface, and recurrent infections may thus be possible. Fourthly, little is known about the role of cellular immune responses to OMPs. The immunodominant T-helper cell epitopes of Hi OMPs have not been characterized. Therefore, the identification of the functional T-helper cell epitopes and the conserved, surface-exposed and/or protective B-cell epitopes of the P1, P2 and P6 proteins is necessary to determine whether these epitopes can elicit immune responses against Hi infection.
Methods for inducing immunity against disease are constantly improving and the current trend is to use smaller and well defined materials as antigens. The objective is to eliminate the potential side-effects of certain native immunogens, while preserving their immunogenicity and ability to confer protection against disease. Recent studies have indicated that immunization of experimental animals with synthetic peptides representing specific regions of viral or bacterial proteins can induce immune responses against the parent proteins, and neutralize their biological functions (refs. 19 to 22). Thus, synthetic peptides are potential candidate antigens for the production of inexpensive and safe vaccines against infectious diseases. Recent progress in fundamental immunology has revealed that good and effective immunogens should contain two distinct functional antigenic determinants (epitopes). One epitope (T-cell epitope) is designed to be presented in the appropriate MHC class II antigen context to the immune system and induce T-helper cell activity. The other epitope (B-cell epitope) must be recognized by a cognate B-cell antigen receptor to elicit antibody production (refs. 23 to 26). Therefore, in order to produce a potent and efficacious synthetic vaccine, both functional T-helper and B-cell epitopes must be included in the synthetic construct.
Synthetic PRP dimer, trimer and tetramer have been synthesized, purified and conjugated to carrier proteins for animal immunogenicity studies (refs. 27, 28). These studies showed that synthetic PRP trimer-protein conjugates in the presence of strong adjuvants such as complete Freund's adjuvant (CFA) could elicit anti-PRP antibody responses in experimental animals.
Instead of using conventional heterologous carrier proteins, our strategy utilizes synthetic peptides containing immunodominant epitopes from Hi OMPs as additional antigens and as carriers for PRP to develop the first generation of fully synthetic PRP-peptide conjugate vaccines with enhanced protective ability and autologous T-cell priming. Such vaccines also have other potential advantages over the existing vaccines in which PRP is conjugated to a foreign protein (diphtheria toxoid (PRP-D), or tetanus toxoid (PRP-T), or CRM197 (HbOC), or OMP of Neisseria menincitidis). Firstly, the use of synthetic Hi vaccines should help reduce the amount of D or T in any future multivalent combined vaccines, thus minimizing the potential risk of hyperimmunization against these carrier proteins. Secondly, PRP may be coupled to a conserved protective epitope to produce a vaccine against both invasive Hi disease and otitis media.
______________________________________ ABBREVIATIONS AND DEFINITIONS ______________________________________ CRM.sub.197 a non-toxic protein antigenically cross- reactive with diphtheria toxin Hi Haemophilus influenzae Hib Haemophilus influenzae type B MAP multiple antigen peptide MBS m-maleimidobenzoyl-N-hydroxysuccinimide OMP outer membrane protein PEG polyethylene glycol monomethyl ether PRP polyriboseribitol phosphate ______________________________________